New genetic-based methods are being developed to map neuronal interconnectivity. These approaches promise to be extremely powerful since they combine the specificity of cell type-specific promoters with the ability of neurotropic viruses to spread trans-synaptically. However, in their current configuration, these methods rely on transgenic mice to selectively drive the expression of either the tracer or the activator. Several laboratories, including ours, have accumulated experience with the rat or with other mammals for which transgenic technologies are currently cumbersome or unavailable. For this reason, in Specific Aim 1 of the present R21 application we propose a novel method for demonstrating retrograde trans-synaptic labeling of pathways in the mammalian brain, which can be freely applied to different species without the need to generate transgenic animals. Neurons will be directly visualized with a virus vector conditionally expressing green fluorescent protein (GFP), thus providing an extremely high signal to noise ratio and facilitating detection of multisynaptic pathways projecting upon the target. The proposed method utilizes two separate virus vectors delivered in two interconnected regions. The anatomical selectivity with the proposed method is based on the specific connections of the neurons of interest. Subsequent developments could also include the use of cell type-specific promoters for added specificity. In Specific Aim 2, we propose to explore the use of recombinant cholera toxin B subunit (CTb) as a viral vector-encoded tracer to expand the toolbox of genetic tracers. CTb is a reliable, sensitive and robust tracer for both anterograde and retrograde studies. Of particular note is the ability of CTb to trace collateral axons derived from the parent retrogradely labeled somata. Very exciting recent studies have also demonstrated the use of wheat germ agglutinin (WGA) as a genetically-encoded tracer. Recombinant CTb could also be used concomitantly with WGA to simultaneously label two pathways. The results of the present R21 will contribute to the development and refinement of strategies for the regulated delivery of recombinant tracers and, in follow-up studies, other reporters such as functional biosensors.